Selection of Allosteric DNAzymes for Theophylline Sensing by Expression-SELEX.

Aptamers are single-stranded DNA or RNA molecules that bind specific ligands with high affinity. DNA aptamers are especially promising due to their enhanced stability and cost-effectiveness, yet their practical use often necessitates coupling with an expression platform to transduce ligand-binding events into detectable signals. To overcome this limitation, we developed Expression-SELEX, an efficient selection method that integrates a DNAzyme-based expression platform directly into the screening process. Previously, we employed this approach to isolate a phenylalanine-responsive allosteric DNAzyme with significant ligand-dependent self-cleavage activity. However, the broader applicability of Expression-SELEX for discovering diverse ligand-dependent DNAzymes and their utility in clinical detection remained unexplored. In this study, we applied Expression-SELEX to identify theophylline-responsive allosteric DNAzymes as functional replacements for traditional RNA aptamers. Our selection yielded 12,880 unique candidates, with all top 10 enriched candidates displaying both significant theophylline-dependent cleavage activity and excellent specificity, allowing effective discrimination between theophylline and caffeine. Although the cleavage products of these ligand-responsive DNAzymes could be detected through PAGE gel analysis as basic biosensors, we enhanced their functionality by incorporating a fluorophore-quencher pair for liquid-phase assays in 96-well plates. The optimized biosensors exhibited excellent performance in reaction buffer containing 1%-5% urine. These findings highlight the versatility of Expression-SELEX for rapidly generating ligand-responsive DNAzymes and biosensors, underscoring their potential for diagnostic and biomedical applications.